Epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase superfamily. EGFR is known to be involved in regulation of cell proliferation, maturation, and differentiation (Carpenter, G. & Cohen, S. J. Biol. Chem. 265, 7709-7712 (1990)).
Binding epidermal growth factor (EGF) to the extracellular domain of EGFR is thought to induce receptor dimerization, which brings the cytoplasmic tyrosine kinase domain of the two receptors into close proximity, resulting in the activation of intrinsic tyrosine kinase receptors in the intracellular domain, followed by the activation of numerous downstream signal pathways (Schlessinger, J. Cell 103, 211-225 (2000)). Besides this activation mechanism, it has been recently demonstrated that a portion of EGFR activated by EGF is translocated to a nucleus so that it might function as a transcription factor to activate genes required for high-level proliferation activities (Lin, S. Y. et al., Nat. Cell Biol. 3, 802-808 (2001)). In the meantime, spontaneous oligomerization accompanied by tyrosine phosphorylation has been reported for oncogenic mutants lacking either a portion or most of the extracellular domain (Haley, J. D. et al., Oncogene 4, 273-283 (1989); Huang, H. S. et al., J. Biol. Chem. 272, 2927-2935 (1997)). The extracellular domain also likely plays a critical role in suppression of ligand-dependent spontaneous oligomerization.
Three homologues of EGFR (ErbB-2, ErbB-3, and ErbB-4) have been identified in humans. Numerous studies have demonstrated that, in addition to homo-dimerization, these EGF ligands also induce a combinational hetero-oligomerization of different pairs of the EGFR family members (Olayioye, M. A. et al., Embo J. 19, 3159-3167 (2000)).
The three-dimensional structure of a human EGF monomer has been analyzed using high-resolution NMR (J. Mol. Biol. (1992) 227, 271-282). As a result, 2 helical segments (Leu8-Tyr13 and Leu47-Glu51) have been discovered. The first segment has been reported to form a major β-sheet via disulfide bridges (Cys6-Cys20 and Cys14-Cyh31), and the second segment has been reported to form a type II turn on the C-terminus of the protein. This helix has been reported to show amphipathic features with Leu47, Trp50, Trp49, and Leu52 on the hydrophobic surface, and Lys48 and Glu51 on the hydrophilic surface. This helix is thought to participate in the formation of a hydrophobic core (Val34, Arg45, and Trp50) in the periphery of Tyr37, which is a conserved residue. In recent years, analytical results achieved using NMR on EGF dimerization have been reported (J. Biol. Chem (2001) 276, 34913-34917). As a result, 3 disulfide bridges (Cys6-Cys20, Cys14-Cys31, and Cys33-Cys42) have been confirmed, and it has been revealed that it consists of an N-domain (residues 1-32) and a C-domain (residues 33-53). The N-domain has been reported to have an irregular N-terminal peptide segment (residues 1-12) and an anti-parallel β-sheet (residues 19-23 and residues 28-32). In addition, the C-domain has been reported to contain a short anti-parallel β-sheet (residues 36-38 and residues 44-46) and a C-terminal segment (residues 48-53).
Furthermore, research has been conducted on mutation of Arg41 and Leu47. As a result, it is known that these residues are essential for the binding of EGF with its receptor, and substitution of arginine with lysine is not allowed (Mol. Cell. Biol (1989) 9, 4083-4086; FEBS Letters (1990) 261, 392-396; FEBS Letters (1990) 271, 47-50; Biochemistry (1991) 30, 8891-8898; Proc. Nat. Acad. Sci., USA (1989) 86, 9836-9840). This indicates that the arginine side chain of EGF (guanidino group) participates in specific interaction with EGFR. In addition, research has been conducted with point mutation, where altered amino acids have been prepared for Ile23, Ala25, Leu26, Ala30, and Asn32, and experiments and studies have been conducted using them, suggesting that each residue may be an amino acid directly interacting with EGFR. However, though amino acid residues important for the interaction are increasingly presumed by point mutation experiments, information needed for industrial application has not yet been obtained in the current situation because the active conformation of the amino acid side chains, the mode of interaction with EGFR, and the active conformation of the EGFR amino acid side chains are unknown.
Regarding EGFR, although efforts have been made to elucidate the crystal structure, such elucidation has not yet been achieved (J. Biol. Chem (1990), 265, 22082-22085; Acta Crystallogr D Biol Crystallogr (1998) 54, 999-1001), and the three-dimensional structure has been merely modeled by homology modeling (Biochim. Biophys. Acta (2001) 1550, 144-152). In this report, the model structure of EGFR has been built using an insulin receptor and a lymphocyte protein-tyrosine kinase as templates. Moreover, there is a report in which IGF-1R (insulin-like growth factor-1 receptor) has been used as a template (Jorissen R. N. et al., Protein Sci. 2000, 9(2), 310-324; WO 99/62955).
The point mutation experiment conducted by causing mutation of Glu367, Gly441, and Glu472 to result in Lys has revealed that mutations of Glu367Lys and Glu472Lys do not affect the binding with ligands. On the other hand, the mutation of Gly441Lys has been reported to significantly reduce the affinity with EGF (Biochemistry (2001) 40, 8930-8939).
Currently, the development of drug candidate compounds targeting EGFR is in progress (Drugs 2000; Vol. 60 Suppl. 1: 15-23, Clinical Cancer Research 2001; Vol. 7: 2958-2970). Furthermore, many low molecular inhibitors suppressing the kinase activity in the intracellular domain of EGFR have been reported (Drugs 2000; Vol. 60 Suppl. 1: 25-32). However, among low molecular weight compounds, neither medicaments that selectively promote activation by binding to EGFR extracellular domain, nor medicaments that selectively inhibit activation have been marketed as drugs. In general, compared with antibodies and recombinant protein pharmaceutical preparations, the agonists and the antagonists of the low molecular weight compounds can be orally administered in many cases, so that they are more useful as drugs. Therefore, lowering the molecular weight of a protein pharmaceutical preparation has been attempted for many diseases. However, the development of such a preparation generally necessitates further trial and error, so that the development of a drug useful for patients always requires a long time and a high cost. Furthermore, regarding an antagonist targeting an intracellular kinase domain, it is predicted that the backbone is limited because the antagonist needs to permeate a cell membrane, and an excessive dose thereof is required. In addition, there are no experimental three-dimensional structures (crystal structures or NMR structures) of EGFR kinase domains, so that many inductions and syntheses on a trial-and-error basis are required for the provision of selectivity. Moreover, antagonists acting on an ATP binding region intramolecularly contain a pharmacophore having ATP-like chemical properties in many cases. Thus, such an antagonist will often be a nucleic acid analogue in terms of the properties of a compound, and future side effects pose difficulties.